Catalytic site inhibition of insulin-degrading enzyme by a small molecule induces glucose intolerance in mice

Insulin-degrading enzyme (IDE) is a protease that cleaves insulin and other bioactive peptides such as amyloid-β. Knockout and genetic studies have linked IDE to Alzheimer's disease and type-2 diabetes. As the major insulin-degrading protease, IDE is a candidate drug target in diabetes. Here we have used kinetic target-guided synthesis to design the first catalytic site inhibitor of IDE suitable for in vivo studies (BDM44768). Crystallographic and small angle X-ray scattering analyses show that it locks IDE in a closed conformation. Among a panel of metalloproteases, BDM44768 selectively inhibits IDE. Acute treatment of mice with BDM44768 increases insulin signalling and surprisingly impairs glucose tolerance in an IDE-dependent manner. These results confirm that IDE is involved in pathways that modulate short-term glucose homeostasis, but casts doubt on the general usefulness of the inhibition of IDE catalytic activity to treat diabetes.

IDE is a well-established protease capable of degrading Aβ 1 as well as the amyloid precursor protein intracellular domain (AICD) 2 . Endogenously secreted Aβ 1-40 is significantly elevated in primary neuronal cultures from ide KO 3 . We thus measured the effect of 1 and 10 on Aβ 1-40 accumulation in cultured human SY5Y neuroblastoma cells. Our ELISA data revealed significant elevations of the peptide in the culture treated with 1, but not with 10, a 100 fold less active analog. This confirms the inhibitory effect of 1 on IDE activity in cell culture. Glucose-Stimulated Insulin Secretion on islets from C57BL/6J wt and Ide-/-mice. Islets isolated from C57BL/6J wild type (a, b) and Ide KO (c, d) were treated successively with 2.8 and 28 mM glucose, in the presence of compound 1 at 3 or 10µM (blue bars) or vehicle (grey bars). The concentrations of insulin (µg/L) in supernatants were then measured after 1 hour incubation time using ELISA. Data are expressed as means of two measurements. Insulin secretion indexes (e) were calculated as the ratio of concentrations of insulin measured at 28mM glucose to the concentrations measured at 2.8 mM for each treatment condition (blue bars for compound 1, grey bars for vehicle). Data shown are mean +/-s.e.m (n=3).       All reagents, solvents and starting materials were purchased from commercial suppliers and used without further purification. Melting points were determined using a Büchi B-540 melting point apparatus and are uncorrected. 1 H NMR spectra were recorded on a Brucker Avance 300 MHz spectrometer with methanol-d6, CDCl 3 or DMSO-d6 as the solvent. 13 C NMR spectra are recorded at 100 MHz. All coupling constants are measured in hertz (Hz) and the chemical shifts (δ) are quoted in parts per million (ppm). Liquid chromatography mass spectroscopy analyses (LC-MS) were performed using LCMS-MS triple-quadrupole system (Waters) with a C 18 TSK-GEL Super ODS (2 µm  High resolution mass spectroscopy (HRMS) were carried out on an Waters LCT Premier XE (TOF), ESI ionization mode, with a Waters XBridge C 18 (150*4.6 mm, 3.5 µm particle size). LCMS gradient starting from 98% ammonium formate buffer 5 mM (pH 9.2) and reaching 95% CH 3 CN / 5% ammonium formate buffer 5 mM (pH 9.2) within 15 min at a flow rate of 1 mL/min was used.

Synthesis of 1-10, 19 (Structures in Supplementary Table 6)
The azide 20a or 20b (1 equiv) and the alkyne (1equiv) were dissolved separately in DMSO (100-150µL) then added to a mixture of tBuOH/water (1/1) or directly solubilised in a mixture of DMF/water (1/1). CuSO 4 :5H 2 O (0.1equiv) and sodium ascorbate (1 equiv) were added. After 12 h of stirring at room temperature, the media was filtered. In most cases, concentration of the filtrate, followed by precipitation in water, filtration and washing with ethyl acetate gave a pure product. If necessary, the final triazole was purified by flash chromatography on silica gel (CH 2 Cl 2 /MeOH).

N-[1-(1-Hydroxycarbamoylmethyl-2-phenyl-ethyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-fluoro-benzamide (19).
White solid ( General procedure for the chemical synthesis of the N-hydroxy amidines 22-23. Aminoacetonitrile hydrochloride (1 eq) was placed in a flask where pyridine was carefully added to obtain a solution, and to this was added acyl chloride (1.05 eq) dropwise over 20 min. After stirring overnight at room temperature, water was carefully added; pyridinium hydrochloride dissolved while the product precipitated as a white solid. The precipitate was collected by filtration and washed with water. If no precipitation was observed, the mixture was extracted three times with dichloromethane. The combined organic layers were dried over MgSO 4 , filtrated and concentrated under reduced pressure to give the cyanomethylbenzamide as a solid. To a solution of the obtained cyanomethylbenzamide (1.0 equiv) in methanol cooled to 0 °C were added hydroxylamine hydrochloride (1.0 equiv) and triethylamine (1.0 equiv), and the mixture was stirred at room temperature or heated to reflux overnight. The mixture was concentrated under reduced pressure. Water was added to the residue and the solid was collected by filtration, washed with water and dried to give the N-hydroxy amidine product as a white amorphous solid.
N-(N-hydroxycarbamimidoylmethyl)-benzamide (  To a solution of 21 (1 eq) in DCM was added carbonyldiimidazole (2.2 eq). The reaction mixture was stirred at room temperature for 10 min. N-hydroxy amidine (1 eq) (22 or 23) was added and the solution was stirred at room temperature for 16 h. The solvent was removed by evaporation in vacuo, and the residue was dissolved in EtOAc and washed twice with water. The organic layer was dried (MgSO 4 ) and the solvent was removed by evaporation in vacuo. The residue was solubilized in DMF and the solution was heated to reflux for 4 h. The reaction mixture was cooled to room temperature, the solvent was removed by evaporation in vacuo, and the residue was dissolved in EtOAc and washed twice with water. The organic layer was dried (MgSO 4 ) and the solvent was removed by evaporation in vacuo. The oil was purified by flash chromatography on silica gel (cyclohexane/EtOAc 1:0 to 1:1 (v/v)) to afford oxadiazole as a colorless oil. The resulting ester was dissolved and EtOH (24 mL) and aqueous NaOH (2 M 5 (v/v)).

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The solvent was evaporated under reduced pressure and the residue was dissolved in CH 2 Cl 2 and washed three times with a 5% NaHCO 3 (aq) solution and once with water.